Disposable glucose test strips, and methods and compositions for making same

ABSTRACT

An improved disposable glucose test strip for use in a test meter of the type which receives a disposable test strip and a sample of blood from a patient and performs an electrochemical analysis is made using a working formulation containing a filler, an enzyme effective to oxidize glucose, e.g., glucose oxidase, and a mediator effective to transfer electrons from the enzyme. The working formulation is printed over a conductive carbon base layer to form a working electrode. The filler, for example a silica filler, is selected to have a balance of hydrophobicity and hydrophilicity such that one drying it forms a two-dimensional network on the surface of the conductive base layer. The response of this test strip is essentially temperature independent over relevant temperature ranges and is substantially insensitive to the hematocrit of the patient.

BACKGROUND OF THE INVENTION

This application relates to disposable glucose test strips for use inelectrochemical determinations of blood glucose, and to methods andcompositions for use in making such strips.

Glucose monitoring is a fact of everyday life for diabetic individuals,and the accuracy of such monitoring can literally mean the differencebetween life and death. To accommodate a normal life style to the needfor frequent monitoring of glucose levels, a number of glucose metersare now available which permit the individual to test the glucose levelin a small amount of blood.

Many of these meters detect glucose in a blood sample electrochemically,by detecting the oxidation of blood glucose using an enzyme such asglucose oxidase provided as part of a disposable, single use electrodesystem. Examples of devices of this type are disclosed in EuropeanPatent No. 0 127 958, and U.S. Pat. Nos. 5,141,868, 5,286,362,5,288,636, and 5,437,999 which are incorporated herein by reference.

In general, existing glucose test strips for use in electrochemicalmeters comprise a substrate, working and reference electrodes formed onthe surface of the substrate, and a means for making connection betweenthe electrodes and the meter. The working electrode is coated with anenzyme capable of oxidizing glucose, and a mediator compound whichtransfers electrons from the enzyme to the electrode resulting in ameasurable current when glucose is present. Representative mediatorcompounds include ferricyanide, metallocene compounds such as ferrocene,quinones, phenazinium salts, redox indicator DCPIP, andimidazole-substituted osmium compounds.

Working electrodes of this type have been formulated in a number ofways. For example, mixtures of conductive carbon, glucose oxidase and amediator have been formulated into a paste or ink and applied to asubstrate. EP 0 127 958 and U.S. Pat. No. 5,286,362. In the case ofdisposable glucose strips, this application is done by screen printingin order to obtain the thin layers suitable for a small flat test strip.The use of screen printing, however, introduces problems to theoperation of the electrode.

Unlike a thicker carbon paste electrode which remains fairly intactduring the measurement, screen printed electrodes formed from carbonpastes or inks are prone to break up on contact with the sample. Theconsequences of this breakup are two-fold. Firstly, the components ofthe electrode formulation are released into solution. Once thesecomponents drift more than a diffusion length away from the underlyingconductive layer, they no longer contribute toward the measurement, butin fact diminish the response by depleting inwardly-diffusing analyte.Secondly, the breakup of the screen printed electrode means that theeffective electrode area is falling over time.

The combination of these two effects results in current transients whichfall rapidly from an initial peak over the period of the measurement,and a high sensitivity to oxygen which quickly competes with themediator for the enzyme. This fact is clearly demonstrated by the muchlower currents measured in blood samples than in plasma samples or otheraqueous media, and can result in erroneous readings. A furtherconsequence is that the transients are often "lumpy" as the electrodebreaks up in a chaotic manner. Lumpy transients either give rise toerroneous readings or rejected strips, neither of which are acceptable.

In addition to the potential for electrode breakup of screen-printedcarbon-based electrodes, known electrodes used in disposable glucosetest strips have been kinetically-controlled, i.e., the current dependson the rate of conversion of glucose by the enzyme. Because the responsemeasured by the instrument represents a balance between the reaction ofenzyme and mediator, enzyme and glucose and enzyme and oxygen, andbecause each of these reactions has its own dependence on temperature,the response of a kinetically-controlled test strip is very sensitive tothe temperature of the sample. Substantial variation in the measuredglucose value can therefore occur as a result of variations in samplehandling.

Because of the importance of obtaining accurate glucose readings to thewell-being of a patient using the meter and disposable test strips, itwould be highly desirable to have a glucose test strip which did notsuffer from these drawbacks, and which therefore provided a moreconsistent and reliable indication of actual blood glucose values,regardless of actual conditions. It is therefore an object of thepresent invention to provide disposable glucose test strips which arenot prone to electrode breakup on contact with a sample.

It is a further object of this invention to provide glucose test stripswhich provide a glucose reading that is essentially independent of thehematocrit of the sample.

It is a further object of the present invention to provide glucose teststrips which are substantially independent of temperature over a rangebetween normal body temperature and room temperature.

It is a further object of the invention to provide test strips whichprovide a substantially flat current transient, without significantdecay for periods of at least 10 seconds after the peak current level isobtained.

SUMMARY OF THE INVENTION

The present invention provides an improved disposable glucose test stripfor use in a test meter of the type which receives a disposable teststrip and a sample of blood from a patient and performs anelectrochemical analysis of the amount of glucose in the sample. Thetest strip comprises:

(a) a substrate;

(b) a reference electrode;

(c) a working electrode; and

(d) means for making an electrical connection between the reference andworking electrode and a glucose test meter. The working electrodecomprises a conductive base layer disposed on the substrate and anon-conductive coating disposed over the conductive base layer. Thenon-conductive coating comprises a filler which has both hydrophobic andhydrophilic surface regions, an enzyme effective to oxidize glucose,e.g., glucose oxidase, and a mediator effective to transfer electronsfrom the enzyme to the conductive base layer. The filler is selected tohave a balance of hydrophobicity and hydrophilicity such that on dryingit forms a two-dimensional network on the surface of the conductive baselayer. Preferred filler are non-conductive silica fillers. The responseof this test strip is dependent on the diffusion rate of glucose, not onthe rate at which the enzyme can oxidize glucose, such that theperformance of the test strip is essentially temperature independentover relevant temperature ranges. Further, the silica appears to form atwo-dimensional network which excludes red blood cells, thus renderingthe test strip substantially insensitive to the hematocrit of thepatient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows an electrode structure useful in a disposable teststrip in accordance with the invention;

FIG. 2 shows a test strip in accordance with the invention;

FIGS. 3A-3C show the current measured as a function of glucoseconcentration for three different hematocrit levels;

FIG. 4 shows the relationship of the glucose-concentration dependence ofthe measured current as a function of hematocrit;

FIGS. 5A-5C show the current measured as a function of glucose in bloodand a control solution for three different variations of the conductivebase layer;

FIG. 6A and 6B show the current measured as a function of glucose at twodifferent temperatures;

FIG. 7 shows a further embodiment of a glucose test strip according tothe invention; and

FIGS. 8A and 8B show a current transients observed using a test stripaccording to the invention and a commercial carbon-based test strip.

DETAILED DESCRIPTION OF THE INVENTION

FIGS. 1A and 1B show electrodes useful in a disposable test strip inaccordance with the invention. As shown, the electrodes are formed on asubstrate 10. On the substrate 10 are placed a conductive base layer 16,a working electrode track 14, a reference electrode track 15, andconductive contacts 11, 12, and 13. An insulating mask 18 is thenformed, leaving a portion of the conductive base layer 16, and thecontacts 11, 12 and 13 exposed. A region of a working coating 17 is thenapplied over the insulating mask 18 to make contact with conductive baselayer 16.

The assembly shown in FIG. 1 provides a fully functional assembly forthe measurement of blood glucose when connected to a meter.Advantageously, however, the electrode strips of the invention arefinished by applying a polyester mesh 21 over the region of the workingcoating 17 of the electrode assembly 22, and then a top cover 23 toprevent splashing of the blood sample. (FIG. 2) The polyester mesh actsto guide the sample to the reference electrode, thereby triggering thedevice and initiating the test.

The substrate 10 used in making the test strips of the invention can beany non-conducting, dimensionally stable material suitable for insertioninto a glucose test meter. Suitable materials include polyester films,for example a 330 micron polyester film, and other insulating substratematerials.

The working electrode track 15, the reference electrode track 14, andconductive contacts 11, and 12 can be formed from essentially anyconductive material including silver, Ag/AgC1, gold, or platinum/carbon.

The conductive base layer 16 is preferably formed from conductivecarbon. Preferred conductive carbon are ERCON ERC1, ERCON ERC2 andAcheson Carbon Electrodag 423. Carbon with these specifications isavailable from Ercon Inc. (Waltham, Mass., USA), or Acheson Colloids,(Princes Rock, Plymouth, England). The conductive base layer 16 makescontact with working electrode track 15, and is close too but notcontacting the end of reference electrode track 15.

The insulating layer 18 can be formed from polyester-based printabledielectric materials such as ERCON R488-B(HV)-B2 Blue.

The key to the performance achieved using the present invention is inthe nature of the coating 17. This coating contains a filler which hasboth hydrophobic and hydrophilic surface regions, an enzyme which canoxidize glucose, and a mediator which can transfer electrons from theenzyme to the underlying conductive base layer 16.

A preferred filler for use in the coating 17 is silica. Silica isavailable in a variety of grades and with a variety of surfacemodifications. While all silica compounds tested resulted in a productwhich could measure glucose under some conditions, the superiorperformance characteristics of glucose test strip of the invention areobtained when a silica having a surface modification to render itpartially hydrophobic is used. Materials of this type include Cab-O-SilTS610, a silica which is modified by partial surface treatment withmethyl dichlorosilane; Cab-o-Sil 530, a silica which is modified by fullsurface treatment with hexamethyl disilazane; Spherisorb C4 silica,which is surface modified with 4 carbon chains; and other similarlymodified by silicas, or combinations thereof. Silica with a surfacemodification which is too hydrophobic should be avoided, however, sinceit has been observed that C18-modified silica is too hydrophobic to forma printable ink.

During the process of manufacturing the ink of the invention, theparticles are broken down by homogenization to expose hydrophilic innerportions of the silica particles. The actual particles present in theink therefore have both hydrophilic and hydrophobic regions. Thehydrophilic regions form hydrogen bonds with each other and with water.

When this material is formulated into an ink as described below inExample 1, and screen printed onto the conductive base layer 16, thedual nature of the material causes it to form layers of two-dimensionalnetworks which take form as a kind of honeycomb. On rehydration, thislayer does not break up, but swells forming a gelled reaction zone inthe vicinity of the underlying conductive base layer 16. Enzyme,mediator and glucose move freely within this zone, but interferingspecies such as red blood cells containing oxygenated hemoglobin areexcluded. This results in a device in which the amount of currentgenerated in response to a given amount of glucose varies by less than10 percent over a hematocrit range of 40 to 60%, and which is thussubstantially insensitive to the hematocrit of the sample, and in factperforms substantially the same in blood as in a cell-free controlsolution. (FIGS. 3A-C, FIG. 4 and FIG. 5A-5C)

Furthermore, the gelled reaction zone presents a greater barrier toentry of glucose which makes the device diffusion, rather thankinetically limited. This leads to a device in which the measuredcurrent varies by less than 10 percent over a temperature range from 20°C. to 37° C. and which is thus essentially temperature independent.(FIGS. 6A and 6B)

The working layer is advantageously formed from an aqueous compositioncontaining 2 to 10% by weight, preferably 4 to 10% and more preferablyabout 4.5% of a binder such as hydroxyethylcellulose or mixtures ofhydroxyethylcellulose with alginate or other thickeners; 3 to 10% byweight, preferably 3 to 5% and more preferably about 4% silica; 8 to 20%by weight, preferably 14 to 18% and more preferably about 16% of amediator such as ferricyanide; and 0.4 to 2% by weight, preferably 1 to2% and more preferably about 1.6% of an enzyme such as glucose oxidase,assuming a specific activity of about 250 units/mg, or about 1000 to5000 units per gram of ink formulation.

The working layer may also include additional ingredients withoutdeparting from the scope of the invention. For example, thenonconducting layer may include an antifoam. In addition, thenonconducting layer may be formulated with a buffering agent to controlthe pH of the reaction zone. The pH may be maintained at a level withinthe range from about pH 3 to pH 10. It is of particular utility tomaintain the pH of the device at a level above 8 because at this pHoxygen bound to hemoglobin is not released. Further, at this pH, thereaction rate of glucose oxidase with oxygen is very low. Thus,selection of an appropriate pH can further stabilize the performance ofthe test strip against the effects of varying hematocrit.

FIG. 7 shows an alternative embodiment of the invention. In thisembodiment, a second working layer 71 is disposed over the first workinglayer 17. This layer is formed from a composition which is identical tothe first working layer except that the enzyme or both the enzyme andthe mediator are omitted. This layer further isolates the conductivebase layer from contact with oxygen-carrying red blood cells, thusreducing the effects of oxygen. Furthermore, to the extent that enzymemay tend to diffuse away from the surface of the electrode during thecourse of the measurement, this layer provides an increased region inwhich it will have mediator available for the transfer of electrons.

EXAMPLE 1

A non-conducting formulation for preparation of the working layer 17 wasmade as follows. 100 ml of 20 mM aqueous trisodium citrate was adjustedto pH 6 by the addition of 0.1M citric acid. To this 6 g of hydroxyethylcellulose (HEC) was added and mixed by homogenization. The mixture wasallowed to stand overnight to allow air bubbles to disperse and thenused as a stock solution for the formulation of the coating composition.

2 grams Cab-o-Sil TS610 silica and 0.1 grams of Dow Corning antifoamcompound was gradually added by hand to 50 grams of the HEC solutionuntil about 4/5 of the total amount has been added. The remainder isadded with mixing by homogenization. The mixture is then cooled for tenminutes in a refrigerator. 8 g of potassium hexacyanoferrate (III) isthen added and mixed until completely dissolved. Finally, 0.8 g ofglucose oxidase enzyme preparation (250 Units/mg) is added and thethoroughly mixed into the solution. The resulting formulation is readyfor printing, or can be stored with refrigeration.

EXAMPLE 2

To prepare glucose test strips using the ink formulation of Example 1, aseries of patterns are used to screen print layers onto a 330 micronpolyester substrate (Melinex 329). The first step is the printing ofcarbon pads. An array of 10×50 pads of carbon is formed on the surfaceof the polyester substrate by printing with EC2 carbon. (Ercon) Theprinted substrate is then passed through a heated dryer, and optionallycured at elevated temperature (e.g. 70° C.) for a period of 1 to 3weeks.

Next, an array of silver/silver chloride connecting tracks and contactsis printed onto the substrate using ERCON R-414 (DPM-68)1.25bioelectrode sensor coating material and dried. One working track whichmakes contact with the carbon pad and one reference track is printed foreach carbon pad in the array.

A dielectric layer is then printed using ERCON R488-B(HV)-B2 Blue anddried. The dielectric layer is printed in a pattern which coverssubstantially all of each devices, leaving only the contacts, the tip ofthe reference electrode and the carbon pads uncovered.

On top of the dielectric layer the ink of Example 1 is used to form aworking layer overlaid on top of each conductive carbon pad.

Polyester mesh strips (Scrynel PET230 HC) are then laid down across thesubstrate in lines, covering the reactions areas exposed by the windowsin the dielectric. A 5 mm wide polyester strip (50 microns thick) isthen applied over the top of the mesh strips, and the edges of theelectrodes are heat sealed. Finally, the substrate is cut up to provide50 individual electrodes, for example having a size of 5.5 mm wide and30 mm long.

EXAMPLE 3

Test strips manufactured using the ink formulation of Example 1 in themanner described in Example 2 were placed in a test meter with anapplied voltage of 500 mV and used to test blood samples having varyingglucose concentrations and hematocrits ranging from 40% to 60%. FIGS.3A-3C show the current measured 25 seconds after applying the voltage asa function of the glucose concentration, and FIG. 4 plots the slope ofthe glucose response as a function of hematocrit. As can be seen, theindicators produce highly reproducible current levels which areessentially independent of hematocrit.

EXAMPLE 4

Glucose test strips in accordance with the invention were made inaccordance with Example 2, except the non-conductive layer was formedwith 7 g Spherisorb C4 and 1 g Cab-o-Sil TS610. This formulation waslaid down on two three different types of carbon-containing conductivebase layers as follows:

A: Ercon EC1

B: Ercon EC2

C: Ercon EC2 on top of Acheson Carbon, Electrodag 423 SS.

These test strips were used to measure varying levels of glucose ineither a control solution (One Touch Control Solution, Lifescan Inc.)containing glucose in an inert solution or in blood at an appliedvoltage of 425 mV. The current observed 25 seconds after the voltage wasapplied was measured. FIGS. 5A-5C show the results obtained for thethree formulations, A, B, and C, respectively. In all cases, the slopeof the line showing the response of the meter to different glucoseconcentrations was essentially the same whether the measurement weremade in blood or the control solution. Thus, this further demonstratesthe independence of the test strips of the invention from the oxygencontent and hematocrit of the sample, as well as the ability to usevaried material as the conductive base layer.

EXAMPLE 5

Test strips prepared in accordance with Example 2 were tested at twodifferent sample temperatures, namely 37° C. and 20° C. using an appliedvoltage of 425 mV. FIGS. 6A and 6B show the current measured 25 secondsafter applying the voltage as a function of glucose concentration. Ascan be seen, the slopes of the two lines are essentially identical(0.1068 at 20° C. versus 0.1009 at 37° C.), thus demonstrating that thetest strips provide essentially temperature-independent behavior over atemperature range from ambient to physiological temperatures.

EXAMPLE 6

The current transient was measured for a test strip prepared inaccordance with Example 2 and for a commercial test strip made with acarbon-containing ink. The results are shown in FIGS. 8A and 8B. Asshown, the test strip of the invention (FIG. 8A) provides a very flattransient which maintains more than 50% of the peak current for a periodof more than 25 seconds after the initial response from the test strip.In contrast, the carbon-based electrode exhibited an almost immediatedecay in the current, having lost 50% of the peak current in a period ofthe first 1 to 2 seconds after the initial response from the test strip.This makes timing of the measurement difficult if peak current valuesare to be captured, or reduces the dynamic range of the meter iscurrents must be measrured after substantial decay has occurred. Thus,the test strips of the invention are advantageous in that they providetest strips in which the amount of current generated in response to agiven amount of glucose decays by less than 50% in the 5 secondsfollowing peak current generation.

We claim:
 1. A disposable glucose test strip for use in a test meter ofthe type which receives a disposable test strip and a sample of bloodfrom a patient and performs an electrochemical analysis of the amount ofglucose in the sample, comprising:(a) a substrate; (b) a referenceelectrode; (c) a working electrode, said working electrode comprising aconductive base layer disposed on the substrate and a first workingcoating disposed over the conductive base layer, said first workingcoating comprising a filler having both hydrophobic and hydrophilicsurface regions such that it forms a network upon drying, an enzymeeffective to oxidize glucose, and a mediator effective to transferelectrons from the enzyme to the conductive base layer; and (d) meansfor making an electrical connection between the reference and workingelectrode and a glucose test meter.
 2. The test strip of claim 1,wherein the working layer is non-conductive.
 3. The test strip of claim2, wherein the filler is silica.
 4. The test strip of claim 3, whereinthe conductive base layer comprises conductive carbon.
 5. The test stripof claim 3, wherein the enzyme is glucose oxidase.
 6. The test stripaccording to claim 3, wherein the mediator is ferricyanide.
 7. The teststrip of claim 3, wherein the first working layer is formed from anaqueous composition comprising weight 2 to 10% by weight of a binder 3to 10% by weight of silica; 8 to 20% by weight of a mediator; and 1000to 5000 units per gram of the aqueous composition of an enzyme foroxidizing glucose.
 8. The test strip of claim 3, wherein the silica is asilica which has been modified by partial surface treatment with methyldichlorosilane.
 9. The test strip of claim 8, wherein the conductivebase layer comprises conductive carbon.
 10. The test strip of claim 8,wherein the enzyme is glucose oxidase.
 11. The test strip of claim 8,wherein the mediator is ferricyanide.
 12. The test strip of claim 8,wherein the first working layer is formed from an aqueous compositioncomprising weight 2 to 10% by weight of a binder 3 to 10% by weight ofsilica; 8 to 20% by weight of a mediator; and 1000 to 5000 units pergram of the aqueous composition of an enzyme for oxidizing glucose. 13.The test strip of claim 3, further comprising a second working layercomprising silica, a binder and a mediator but no glucose-oxidizingenzyme.
 14. The test strip of claim 3, further comprising a secondworking layer comprising silica and a binder but no glucose-oxidizingenzyme.
 15. The test strip of claim 1, further comprising a secondworking layer comprising a filler, a binder and a mediator but noglucose-oxidizing enzyme.
 16. The test strip of claim 1, furthercomprising a second working layer comprising a filler and a binder butno glucose-oxidizing enzyme.
 17. An aqueous composition comprising abinder, a silica filler having both hydrophobic and hydrophilic surfaceregions, at least one of an enzyme effective to oxidize glucose and anelectron transfer mediator.
 18. The composition of claim 17, wherein thefiller is non-conductive.
 19. An aqueous composition comprising 2 to 10%by weight of a binder; 3 to 10% by weight of silica; 8 to 20% by weightof a mediator; and 1000 to 5000 units per gram of the aqueouscomposition of an enzyme for oxidizing glucose.
 20. The composition ofclaim 19, wherein the silica has both hydrophobic and hydrophilicsurface regions.
 21. The composition of claim 20, wherein the binder ishydroxyethylcellulose.
 22. The composition of claim 19, wherein theenzyme is glucose oxidase.
 23. The composition of claim 19, wherein themediator is ferricyanide.
 24. A method for making a disposable teststrip for the electrochemical detection of glucose, comprising the stepsof:(a) applying working and reference electrode tracks to a substrate;(b) applying a conductive base layer in contact with the workingelectrode track; and (c) applying a working layer over the conductivebase layer, wherein the working layer comprising a filler having bothhydrophobic and hydrophilic surface regions such that it forms a networkupon drying, an enzyme effective to oxidize glucose, and a mediatoreffective to transfer electrons from the enzyme to the conductive baselayer.
 25. The method of claim 24, wherein the filler is non-conductive.26. The method of claim 25, wherein the filler is silica.
 27. The methodof claim 26, wherein the conductive base layer comprises conductivecarbon.
 28. The method of claim 26, wherein the enzyme is glucoseoxidase.
 29. The method of claim 26, wherein the mediator isferricyanide.
 30. The method of claim 26, wherein the first workinglayer is formed from an aqueous composition comprising weight 2 to 10%by weight of a binder 3 to 10% by weight of silica; 8 to 20% by weightof a mediator; and 1000 to 5000 units per gram of the aqueouscomposition of an enzyme for oxidizing glucose.
 31. The method of claim30, wherein the silica is a silica which has been modified by partialsurface treatment with methyl dichlorosilane.
 32. A disposable glucosetest strip comprising first and second electrodes which produce acurrent indicative of the amount of glucose in a sample applied to thestrip in response to an applied voltage, wherein the amount of currentgenerated by the electrodes in response to a given amount of glucosevaries by less than 10 percent over a temperature range from 20° C. to37° C.
 33. A disposable glucose test strip comprising first and secondelectrodes which produce a current indicative of the amount of glucosein a sample applied to the strip in response to an applied voltage,wherein the amount of current generated by the electrodes in response toa given amount of glucose varies by less than 10 percent over ahematocrit range of 0 to 60%.
 34. A disposable glucose test stripcomprising first and second electrodes which produces a currentindicative of the amount of glucose in a sample applied to the strip inresponse to an applied voltage, wherein the amount of current generatedby the electrodes in response to a given amount of glucose decays byless than 50% in the 5 seconds following peak current generation.